Tweaking innate immunity: The promise of innate immunologicals …downloads.hindawi.com/journals/cjidmm/2006/195957.pdf · 2019-08-01 · Can J Infect Dis Med Microbiol Vol 17 No

Can J Infect Dis Med Microbiol Vol 17 No 5 September/October 2006 307

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, OntarioCorrespondence: Dr Kenneth L Rosenthal, Centre for Gene Therapeutics, McMaster University, MDCL 4019, 1200 Main Street West,

Hamilton, Ontario L8N 3Z5. Telephone 905-525-9140 ext 22375, fax 905-522-6750, e-mail [email protected]

©2006 Pulsus Group Inc. All rights reserved

AMMI CANADA ANNUAL MEETING SYMPOSIUM

Tweaking innate immunity: The promise of innateimmunologicals as anti-infectives

Kenneth L Rosenthal PhD

KL Rosenthal. Tweaking innate immunity: The promise of

innate immunologicals as anti-infectives. Can J Infect Dis Med

Microbiol 2006;17(5):307-314.

New and exciting insights into the importance of the innate immunesystem are revolutionizing our understanding of immune defenseagainst infections, pathogenesis, and the treatment and prevention ofinfectious diseases. The innate immune system uses multiple familiesof germline-encoded pattern recognition receptors (PRRs) to detectinfection and trigger a variety of antimicrobial defense mechanisms.PRRs are evolutionarily highly conserved and serve to detect infec-tion by recognizing pathogen-associated molecular patterns that areunique to microorganisms and essential for their survival. Toll-likereceptors (TLRs) are transmembrane signalling receptors that activategene expression programs that result in the production of proinflam-matory cytokines and chemokines, type I interferons and antimicro-bial factors. Furthermore, TLR activation facilitates and guidesactivation of adaptive immune responses through the activation ofdendritic cells. TLRs are localized on the cell surface and inendosomal/lysosomal compartments, where they detect bacterial andviral infections. In contrast, nucleotide-binding oligomerizationdomain proteins and RNA helicases are located in the cell cytoplasm,where they serve as intracellular PRRs to detect cytoplasmic infec-tions, particularly viruses. Due to their ability to enhance innateimmune responses, novel strategies to use ligands, synthetic agonistsor antagonists of PRRs (also known as ‘innate immunologicals’) canbe used as stand-alone agents to provide immediate protection ortreatment against bacterial, viral or parasitic infections. Furthermore,the newly appreciated importance of innate immunity in initiatingand shaping adaptive immune responses is contributing to our under-standing of vaccine adjuvants and promises to lead to improved next-generation vaccines.

Key Words: Anti-infectives; CpG DNA; Innate immunity;

Microbicide; NOD1; NOD2; Pathogen-associated molecular patterns;

Pattern recognition receptors; RNA helicases; Toll-like receptors;

Vaccine adjuvant

La modification de l’immunité innée : La promesse des adjuvants immunologiquescomme anti-infectieux

De nouveaux aperçus passionnants de l’importance du systèmeimmunitaire inné révolutionnent notre compréhension de la défenseimmunologique contre les infections, de la pathogenèse, du traitement etde la prévention des maladies infectieuses. Le système immunitaire innéfait appel à de multiples familles de récepteurs de reconnaissance desformes (RRF) codés dans les cellules germinales pour déceler l’infection etdéclencher divers mécanismes de défense antimicrobienne. Les RRF sonthautement conservés pendant leur évolution, et ils permettent de décelerl’infection en reconnaissant les motifs moléculaires propres auxpathogènes uniques aux microorganismes et essentiels à leur survie. Lesrécepteurs Toll sont des récepteurs de signalisation transmembranaire quiactivent les programmes d’expression génique, ce qui entraîne laproduction de cytokines et de chémokines pro-inflammatoires,d’interférons de type I et de facteurs antimicrobiens. De plus, l’activationdes récepteurs Toll facilite et oriente l’activation des réponsesimmunitaires adaptatives par l’entremise de l’activation des cellulesdendritiques. Les récepteurs Toll se trouvent à la surface des cellules etdans les compartiments endosomaux et lysosomaux, où ils détectent lesinfections bactériennes et virales. Par contre, les protéines du domaine del’oligomérisation liant les nucléotides, de même que les hélicases del’ARN, sont sises dans le cytoplasme des cellules, où elles servent de RRFintracellulaires pour dépister les infections cytoplasmiques, notamment lesvirus. Étant donné leur capacité de stimuler les réponses immunitairesinnées, il est possible d’utiliser de nouvelles stratégies visant l’utilisationdes ligands, des agonistes ou des antagonistes synthétiques des RRF(également désignés « adjuvants immunologiques innés ») à titre d’agentsautonomes pour assurer une protection ou un traitement immédiat contreles infections bactériennes, virales ou parasitiques. De plus, la nouvelleappréhension de l’importance de l’immunité innée pour initier et modelerles réponses immunitaires adaptatives contribue à comprendre lesadjuvants vaccinaux et promet de donner lieu à une prochaine générationde vaccins améliorés.

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Despite the fact that the development of antibiotics, vac-cines and, more recently, antivirals has led to the view

that infectious diseases might be easily controlled or even erad-icated, infections remain a constant threat to the health ofdeveloping and developed nations. We are living in a timewhen the world is experiencing the worst pandemic in humanhistory – the HIV pandemic. In 2003, severe acute respiratorysyndrome quickly encircled the globe to infect 8400 people,with a rapidity dramatized by a 78-year-old woman carrying theinfection from Hong Kong to Toronto and precipitating achain reaction that caused 44 deaths in Canada. Twenty well-known diseases, including tuberculosis (TB), malaria andcholera, have re-emerged or spread geographically, often inmore virulent and drug-resistant forms. Collectively,HIV/AIDS, malaria and TB kill six million people per year. Atleast 30 previously unknown agents have been identified since1973, including HIV, hepatitis C virus and Nipah virus, forwhich no cures are available. Importantly, recent concernsthat the H5N1 avian influenza virus could rapidly cause any-where between four and 150 million deaths in a short time areserving as a wake-up call that we, as a global society, must safe-guard ourselves against an increasing number of infectiousthreats. New and exciting insights into activation and theimportance of the innate immune system are revolutionizingour understanding of protection against infections and howvaccine adjuvants work. Based on this new understanding, it isincreasingly being appreciated that enhancement or modula-tion of innate immunity through the use of ‘innate immuno-logicals’ holds great promise as an alternative strategy for therapid control of infections and improved next-generation vac-cines for longer-term control of specific infections.

SENSING INFECTION: THE IMPORTANCE

OF INNATE IMMUNITYThe mammalian immune system has evolved multiple, layeredand interactive defensive systems to protect against infections.These have been broadly divided into innate immunity andadaptive immunity. Innate immunity is the first line of defenseagainst microbial pathogens and acts almost immediately tolimit early proliferation and spread of infectious agents throughthe activation of phagocytic and antigen-presenting cells, such asdendritic cells (DCs) and macrophages, and the initiation ofinflammatory responses through the release of a variety ofcytokines, chemokines and antimicrobial factors, such as inter-ferons (IFNs) and defensins. Innate immunity is evolutionarilyancient, and for many years its study was largely ignored byimmunologists as relatively nonspecific. For the most part,though, we are protected against infection by our innate immunesystem. If infectious organisms penetrate these innate immunedefenses, then our innate defenses facilitate and guide the gener-ation of adaptive immune responses, which are directed againsthighly specific determinants that are uniquely expressed by theinvading pathogen. These responses are dependent on therearrangement of specific antigen-receptor genes in B cells andT cells and result in the production of high-affinity, antigen-specific antibodies (humoral immunity) and T cells (also knownas cell-mediated immunity). In general, antibodies facilitate theremoval, destruction or neutralization of extracellular pathogensand their toxins, whereas T cell-mediated immune responseshelp eliminate or control intracellular pathogens. In contrastwith innate immune responses, adaptive immune responses havethe hallmark of specific immune memory.

There are a number of key questions that have, untilrecently, been unanswered: how does the host innate immunesystem detect infection, and how does it discriminate betweenself and infectious nonself (pathogens)? The recent discoveryand characterization of the toll-like receptor (TLR) family hasprovided great insight into innate immune recognition andestablished a key role of the innate immune system in hostdefense against infection (1-5). The innate immune systemuses multiple families of germline-encoded pattern recognitionreceptors (PRRs) to detect infection and trigger a variety ofantimicrobial defense mechanisms (6). These PRRs are evolu-tionarily highly conserved among species, ranging from plantsand fruit flies to mammals. The strategy of innate immunerecognition is based on the detection of highly conserved andessential structures present in many types of microorganismsand absent from host cells (7,8). Because the targets of innateimmune recognition are conserved molecular patterns, theyare called pathogen-associated molecular patterns (PAMPs).PAMPs have three important features that make them idealtargets for innate immune sensing. First, PAMPs are producedonly by microorganisms and not by host cells. This is the basisfor the discrimination between self and infectious nonself.Second, PAMPs are conserved among microorganisms of a givenclass. This allows a limited number of PRRs to detect the pres-ence of a large class of invading pathogens. For example, a pat-tern in lipopolysaccharide (LPS) allows a single PRR to detectthe presence of any Gram-negative bacteria. Third, PAMPs areessential for microbial survival, and any mutation or loss ofPAMPs are either lethal for the organism or greatly reducestheir adaptive fitness. These new insights into innate immunerecognition are revolutionizing our understanding of immunedefense, pathogenesis, and the treatment and prevention ofinfectious diseases.

WE STAND ON GUARD FOR THEE:

TOLL-LIKE RECEPTORSTLRs represent one family of PRRs that are evolutionarilyconserved transmembrane receptors that detect PAMPs andfunction as signalling receptors. TLRs were first discovered inDrosophila, where they play a role in the development of theventral/dorsal orientation of fruit flies (9). When this gene wasmutated, the flies that developed were found to be ‘toll’, whichis German slang for ‘crazy’ or ‘far out’. Furthermore, flies withmutation of Tolls were found to be highly susceptible to fungalinfections (10). To date, 11 TLRs have been identified inmammals, each sensing a different set of microbial stimuli andactivating distinct signalling pathways and transcription fac-tors that drive specific responses against the pathogens (11).TLRs are type I integral membrane glycoproteins characterizedby extracellular domains containing various numbers ofleucine-rich repeat (LRR) motifs, a transmembrane domainand a cytoplasmic signalling domain homologous to that of theinterleukin-1 receptor (IL-1R), which is termed the Toll/IL-1Rhomology (TIR) domain (12). The LRR domains are com-posed of 19 to 25 tandem LRR motifs, each of which is 24 to29 amino acids in length.

TLR4, the first mammalian TLR discovered, proved to bethe long sought-after receptor for Gram-negative bacterial LPS(13,14). TLR2 recognizes peptidoglycan, in addition to thelipoproteins and lipopeptides of Gram-positive bacteria andmycoplasma (15,16). TLR2 can form heterodimers with TLR1or TLR6 to discriminate between diacyl and triacyl lipopeptides,

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Can J Infect Dis Med Microbiol Vol 17 No 5 September/October 2006308


respectively (15). Furthermore, TLR2, in collaboration withthe non-TLR receptor dectin-1, mediates the response tozymosan, which is found in the yeast cell wall (17). TLR5 rec-ognizes flagellin, a protein component of bacterial flagella(18). TLR11, a close relative of TLR5, was found to be abun-dantly expressed in the urogenital tract of mice and associatedwith protection against uropathogenic bacteria (19); it wasalso recently shown to recognize profilin-like protein from theprotozoan parasite Toxoplasma gondii (20). TLR3, 7, 8 and 9recognize nucleic acids and are not expressed on the cell sur-face, but are exclusively expressed in endosomal compartments(21,22). TLR3 is involved in the recognition of double-strandedRNA (dsRNA) generated during viral infection (23), whereasthe closely related TLR7 and TLR8 recognize viral single-stranded RNA rich in guanosine and uridine (24,25) and syn-thetic imidazoquinoline-like molecules imiquimod (R-837) andresiquimod (R-848) (26,27). TLR9 mediates the recognitionof bacterial and viral unmethylated CpG DNA motifs (28) andwas recently also shown to recognize non-DNA pathogeniccomponents, such as hemozoin from malarial parasites (29).

TLRs can also be divided into six major subfamilies basedon sequence similarity (30), each recognizing related PAMPS.The subfamily consisting of TLR1, TLR2 and TLR6 recognizeslipopeptides; TLR3 recognizes dsRNA; TLR4 recognizes LPS;TLR5 recognizes flagellin; the TLR9 subfamily, which includeshighly related TLR7 and TLR8, recognizes nucleic acids; andthe PAMP for the remaining family, TLR11, is unknown.Importantly, the subcellular localization of TLRs correlateswith the nature of their ligands, rather than sequence similarity(2). TLR1, 2, 4, 5 and 6 are present on the surface plasmamembrane, where they recognize bacterial and viral compo-nents, while antiviral TLRs, including TLR3, 7, 8 and 9, areexpressed in intracellular endosomes. Because nucleic acids rec-ognized by antiviral TLRs are also found in vertebrates, theirlocation in endosomes limits their reactivity to self nucleicacids (31).

Signalling by TLRs is complex and has been reviewed else-where (12,32). Briefly, all TLRs, with the exception of TLR3,signal through the adaptor molecule myeloid differentiationfactor 88, a cytoplasmic protein containing a TIR domain anda death domain. Ultimately, nuclear factor-κB and mitogen-activated protein kinases are activated downstream of tumournecrosis factor receptor-associated factor 6, leading to the pro-duction of proinflammatory cytokines and chemokines, such astumor necrosis factor-alpha, IL-6, IL-1β and IL-12. In additionto myeloid differentiation factor 88, TLR3 and TLR4 signalthrough TRIF, another TIR-containing adaptor that isrequired for the production of type I interferons and type Iinterferon-dependent genes.

TLRs are expressed on a variety of immune and non-immune cells. Murine macrophages express TLR1 through 9,reflecting their importance in the initiation of proinflammatoryresponses. Plasmacytoid DCs (pDCs) that produce largeamounts of type I interferons during viral infections expressTLR7 and 9. All conventional DCs in the mouse expressTLR1, 2, 4, 6, 8 and 9, while TLR3 is confined to the CD8+and CD4-CD8-DC subset (33). In humans, TLR9 expression isrestricted to pDCs and B cells (34,35). TLR3 is not expressedin pDCs of mice or humans. In addition to the focus of expres-sion on immune cells, there is great interest in understandingthe expression of TLRs on mucosal epithelial cells (ECs) thatserve as the first line of defense against most infections. Studies

by Xiao-Dan Yao and colleagues (36) have concentrated onunderstanding expression and regulation of TLRs on ECs inthe genital tract of mice and humans. Most recently, they usedlaser capture microdissection to show that the estrous cycle infemale mice profoundly influences expression of TLRs in thevaginal epithelium. Their findings showed that messengerRNA expression of essentially all TLRs, except TLR11, weresignificantly increased during diestrus and especially followingtreatment with the long-acting progestin Depo-Provera (PfizerInc, USA) (36). These findings should contribute to theunderstanding of innate immune defense against sexuallytransmitted infections (STIs) and enhance the quality ofwomen’s reproductive health.

TLRs induce a range of responses depending on the celltype in which they are activated (33,37). For example, treat-ment of DCs with CpG DNA that acts through TLR9 acti-vates the DCs to mature, including the upregulation ofmajor histocompatibility complex class II and costimulatorymolecules, the production of proinflammatory cytokines andchemokines, and the enhancement of antigen presentation.Similarly, treatment of B cells with CpG induces their acti-vation and proliferation, the secretion of antibody, IL-6 andIL-10, and the resistance of B cells to apoptosis. Activationof immune cells via CpG DNA induces a T helper (Th) type 1-dominated response.

CYTOPLASMIC PATHOGEN

RECOGNITION RECEPTORS

Although TLRs recognize PAMPs at the cell surface or inendosomal/lysosomal membranes, a number of pathogensinvade the cytosol. These pathogens are detected by cytoplas-mic PRRs. Recently, two families of these cytoplasmic recep-tors have been cloned, including the nucleotide-bindingoligomerization domain (NOD)-LRR proteins (38,39) andcaspase-activation recruitment domain (CARD)-helicase pro-teins (40).

NOD-LRR proteins are implicated in innate immunerecognition of specific motifs in bacterial peptidoglycan cellwall components. NOD1 and NOD2 are well studied membersof this large family, which consists of over 20 proteins (38,39).NOD1 and NOD2 proteins possess a C-terminal LRR domainthat mediates ligand sensing, a centrally located NOD thatmediates self-oligomerization, and an N-terminal CARD fortransmitting signals. NOD1 detects gamma-D-glutamyl-meso-diaminopimelic acid (41,42) and NOD2 detectsmuramyl dipeptide (43,44) found in bacterial peptidoglycan.Following ligand binding to NOD1 or NOD2, they oligomer-ize, which results in nuclear factor-κB activation through therecruitment of the serine/threonine kinase RIP2/RICK viaCARD:CARD interactions. NOD1 is expressed in most tis-sues, while NOD2 is limited to monocyte and granulocyte lin-eage and ECs (45-47). These NOD proteins synergize witheach other and with TLRs in activating innate immuneresponses to invasive bacterial pathogens, especially inflamma-tory responses. A mutation in human NOD2 is correlated withsusceptibility to Crohn’s disease, an inflammatory bowel dis-ease (48,49). Thus, Crohn’s disease may be the result of defi-cient innate immune defenses against bacterial infections inthe gut. NOD1 participates in defense against Helicobacterpylori infection (50).

It has been increasingly recognized that there are TLR-independent mechanisms of sensing actively replicating viruses

Innate immunologicals as anti-infectives



in the cytoplasm of infected cells through dsRNA. Indeed,most virus-infected cells secrete type I IFNs in a TLR3-independent manner. CARD-helicase proteins were recentlyidentified as detectors of cytoplasmic viral dsRNA (40).Retinoic acid-inducible protein-1 (RIG-1) and melanomadifferentiation associated gene 5 (MDA-5) are intracellularRNA helicases that contain CARD domains and localize tothe cytoplasm (3). These proteins are widely expressed andupregulated following positive feedback with type I IFNs (40).They recognize intracellular dsRNA and induce IFN-βthrough the activation of IRF3 by TBK1 and IKK1. RIG-1 andMDA-5 protect all cells once they have been infected withvirus, while antiviral TLRs, including TLR7, 8 and 9 on spe-cialized pDCs serve as sentinels of viral infection independentof viral tropism (51). Hepatitis C virus and paramyxovirusesproduce proteins that interfere with activation of RIG-1 orMDA-5, respectively (52,53). Thus, two innate antiviral path-ways trigger type I IFNs in response to dsRNA. RNA virusesreplicating in the cytoplasm are recognized by RIG-1, whereasTLR3 has been suggested to be responsible for recognizingdsRNA contained in apoptotic bodies of virus-infected cellstaken up by DCs (3,5). Overall, the various PRR families thathave been recently identified serve to protect us by sensinginfection at the cell surface, in endosomes/lysosomes inside ofcells, and in the cytoplasm of host cells.

INNATE IMMUNOLOGICALS AS

ANTI-INFECTIVESThe mechanisms by which PRRs mediate host defense againstpathogens are the focus of intense research. Due to their abilityto enhance innate immune responses, novel strategies to useligands, synthetic agonists or antagonists of PRRs (also knownas ‘innate immunologicals’) can be used as stand-alone agentsto provide protection or treatment against infection withintracellular bacteria, parasites and viruses.

One of the earliest models tested examined whether CpGDNA, which acts through TLR9, could redirect a curative Th1response in Balb/c mice infected with Leishmania major, a modelfor a lethal Th2-driven disease (54-56). Treatment of micewith CpG-induced resistance that was associated with IL-12and IFN-γ production. Interestingly, CpG DNA cured micewhen delivered as late as 20 days after lethal L major infection,indicating that CpG can shift an established Th2 response to aprotective Th1 response.

Similarly, mice treated with CpG DNA or synthetic CpGoligodeoxynucleotides (ODNs) were shown to resist infectionfrom high doses of bacteria that cause anthrax, listeria (57),tularemia (58) and TB (59); from viruses such as herpes sim-plex virus (HSV) (60-62), cytomegalovirus and the biothreatagent Ebola virus (63); and from the parasites responsible formalaria and leishmaniasis.

Among infectious diseases, TB and malaria are among theleading causes of death worldwide. The problems associatedwith these infections have worsened due to the emergence ofmultidrug-resistant TB and the resistance of parasites to anti-malarial drugs. Treatment of mice with CpG DNA one or twodays before challenge with Plasmodium yoelii conferred sterileprotection against infection (64). The protection was depend-ent on IL-12 and IFN-γ. Similarly, the treatment of mice withCpG at the time of, or two weeks after, intranasal infectionwith Mycobacterium tuberculosis enhanced survival and reducedmycobacterial outgrowth for up to five weeks in the lungs (59).

This was associated with a decrease in pulmonary inflamma-tion and increased IFN-γ and decreased IL-4 in the lung.

The importance of augmentation of innate immunity byCpG was further demonstrated in a model of acute polymicro-bial sepsis. Treatment of mice with CpG DNA six days beforeinduction of acute polymicrobial peritonitis resulted inincreased resistance to sepsis (65). This resistance was associ-ated with augmented accumulation of neutrophils at the pri-mary site of infection and enhanced antimicrobial activity ofthese cells. Thus, CpG may serve as a potent anti-infective forthe treatment of sepsis. Alternatively, TLR2 or TLR4 antago-nists may prove therapeutically useful in sepsis (66). A natu-rally occurring soluble form of TLR2 (sTLR2) has beenidentified from human blood monocytes (67). sTLR2 binds tosoluble CD14 and limits tumour necrosis factor and IL-8 pro-duction in the presence of bacterial lipopeptides. Thus, sTLR2may modify the endotoxin response. Similarly, a specific TLR4antagonist, eritoran (E 5564), is now being studied in a double-blind, placebo-controlled, human clinical trial to determine itsability to block the toxic effects of endotoxin in healthy vol-unteers (68,69).

Although the natural ligands for TLR7 and TLR8 wererecently identified as single-stranded RNA (25), small moleculeagonists for TLR7 and 8 were already in use clinically againstpapillomavirus-induced genital warts. The imidazoquinoline-like molecules imiquimod and resiquimod are effectiveimmunoadjuvants in eliminating genital warts (70). Now thatthe receptors for these synthetic compounds have been identi-fied, it is possible that improved agonists will be developed.

Importantly, CpG ODNs have also been shown to improvethe ability of immunosuppressed, pregnant and newborn ani-mals to resist infection (71). For example, studies of pregnantmice show that CpG ODN treatment significantly increasedresistance to infection by bacteria and reduced transplacentaltransmission of pathogen from mother to fetus (72).

INNATE IMMUNOLOGICALS AS MUCOSAL

MICROBICIDESMicrobicides are topical compounds delivered to the vaginaland/or rectal mucosa that may prevent STIs (73). Indeed, theyhave been touted as a means of female-controlled HIV preven-tion for over a decade. However, the current strategies of micro-bicides in clinical trials are primarily based on substances thatproduce physical barriers or are based on the use of antiretrovi-ral drugs incorporated into gels, creams or various delivery sys-tems. These drug-based approaches are susceptible not only tothe induction of local inflammation, but also to the same oldproblems encountered with the overuse of antibiotics.

More recent studies have demonstrated that a single dose ofCpG ODN delivered transmucosally to the vaginal mucosa, inthe absence of any viral antigen, protects against genital infec-tion with lethal doses of HSV-2 (60). This protection was medi-ated by the innate immune system because it occurred inknockout mice lacking B cells and T cells. Local intravaginal(IVAG) delivery of CpG ODN resulted in rapid proliferationand thickening of the vaginal epithelium and induction of aTLR9-dependent antiviral state that did not block virus entrybut inhibited viral replication in vaginal epithelial cells (60).More recently, it was determined that mucosal delivery ofdsRNA, the ligand for TLR3, protected against genital HSV-2infection without the local or systemic inflammation seen withCpG ODN (74). Therefore, local delivery of TLR3 ligand may

Rosenthal



be a safer means of protecting against genital viral infection. Itis also highly likely that this innate immune-mediated antiviralstate will protect against a variety of viral STIs, including HIV-1.Thus, topical application of these ‘innate immunologicals’ mayrapidly activate our innate mucosal immune system and inducea local antiviral state, which protects mucosal surfaces againstinfection with sexually transmitted agents. Indeed, thisapproach may be safer and not susceptible to selection of resist-ant pathogens because it would use more ‘natural microbicides’,as well as evolutionarily ancient innate mucosal immuneresponses, for protection.

TAKE THE TOLLWAY: INNATE

IMMUNOLOGICALS AS VACCINE ADJUVANTSMost vaccines, with the exception of those made with repli-cating viruses, require an adjuvant to induce effective immuneresponses. Adjuvants, sometimes called ‘the immunologist’sdirty little secret’, are defined as substances and formulationsthat increase immune responses to an antigen (75). They canalso control the type of immune response elicited and, from apractical standpoint, can reduce the amount of antigen neededin a vaccine. Until recently, adjuvant research has been mostlyempirical and lacking simplifying concepts of how these com-pounds work.

New insights into innate immunity have revolutionizedour understanding of immune activation and the mechanismsunderlying adjuvant activity. Activation of innate immunitythrough PRRs, such as the TLRs, induces a diverse array ofantimicrobial molecules and effector cells, which attackmicroorganisms at multiple levels. Activation of innateimmunity also leads to the release of cytokines andchemokines that recruit and activate many types of cells,including DCs and antigen-presenting cells, which are impor-tant for the transition from innate to adaptive responses.Thus, the activation of the innate immune system throughPRRs using their respective ligands or agonists represents astrategy to enhance immune responses against pathogens,making PRRs excellent adjuvants.

In addition to enhancing innate immunity, TLR ligandsand agonists have been well documented to enhance antigen-specific immune responses (76-82). For example, the ability ofCpG to act as a potent adjuvant was confirmed in studies usingmodel antigens, such as ovalbumin (79), and antigens frominfectious agents, such as hepatitis B virus (77), influenza (83)and HSV (78). Furthermore, these studies showed that CpGserves as a potent adjuvant for both parenteral and mucosalvaccines (80). Although most of these studies made use ofsmall animal models, CpG was also shown to markedlyenhance the response of orangutans to hepatitis B immuniza-tion (84). Orangutans are normally hyporesponsive to currentcommercial hepatitis B vaccines. After two doses, however,100% of animals immunized with vaccine plus CpG had pro-tective levels of antibodies compared with only 8% of animalsimmunized with vaccine alone.

Similarly, in the first human trial of CpG, two weeks afterthe first immunization of normal volunteers, 92% of the sub-jects receiving CpG combined with vaccine had antibodiescompared with 0% of the subjects receiving vaccine alone.Two and four weeks after the second dose, antibody titres weremore than 30-fold higher in subjects receiving vaccine plusCpG versus vaccine alone (85). In a second double-blind study(86), CpG ODNs coadministered with the Fluarix

(GlaxoSmithKline, USA) influenza virus vaccine did notincrease the antibody titre in naive recipients compared withvaccine alone, but did increase antibody levels in subjects withpre-existing influenza virus-specific antibodies. Furthermore,following in vitro restimulation, peripheral blood mononuclearcells from individuals immunized with vaccine plus CpGODNs produced higher levels of IFN-γ than did control vac-cines (86). No serious adverse events attributed to the use ofCpG ODNs were observed.

It has been demonstrated that CpG ODN, a ligand forTLR9, serves as an effective adjuvant for mucosal vaccines(37). Gallichan et al (78) showed that intranasal administra-tion of purified envelope glycoprotein (gB) from HSV-2 plusCpG ODN as an adjuvant induces strong gB-specificimmunoglobulin (Ig) A and IgG in the vaginal tract (persist-ing throughout the estrous cycle), as well as systemic andgenital gB-specific cytotoxic T lymphocytes, and protectsagainst lethal IVAG HSV-2 infection. Subsequently, Dumaiset al (87) showed that intranasal immunization with inacti-vated gp120-depleted HIV-1 plus CpG ODN induces anti-HIV IgA in the genital tract and HIV-specificT cell-mediated immune responses, including the productionof IFN-γ and beta-chemokines. Furthermore, mice immu-nized intranasally with HIV-1 plus CpG induced CD8+T cells in the genital tract, providing cross-clade protectionagainst IVAG challenge with recombinant vaccinia virusesexpressing HIV-1 Gag from different clades (88). Morerecently, although the genital tract has been considered to bea poor immune inductive site, especially following immuniza-tion with nonreplicating antigens, Kwant and Rosenthal (89)showed that IVAG immunization of female mice with recom-binant subunit HSV-2 gB plus CpG induced higher levels ofgB-specific IgG and IgA antibodies in serum and vaginalwashes versus mice immunized with antigen alone, and thatmice immunized with gB plus CpG were better protectedagainst vaginal infection with HSV-2. Thus, it is possible toinduce protective immune responses following IVAG immu-nization with a nonreplicating subunit protein antigen, pro-vided an appropriate mucosal adjuvant is used.

Similarly, TLR4 agonists, such as monophosphoryl lipid A(a chemically modified LPS that retains many of the immunos-timulatory properties of LPS without its toxic effects), is alsoan effective mucosal adjuvant (90). Mice immunizedintranasally with influenza hemagglutinin plus MPL (clinicalgrade formulation of monophosphoryl lipid A) generatedmucosal immune responses, including IgA in vaginal washes,and enhanced protection against intranasal challenge withinfluenza virus. Novel approaches to vaccine developmentcould exploit the effects of TLR-activating compounds oninnate and adaptive immune responses, especially at mucosalsurfaces.

In a novel approach to understand what makes ‘successful’vaccines, Bali Pulendran and his associates recently examinedyellow fever virus 17D (YF-17D) vaccine. The YF-17D vac-cine has been administered to over 400 million people world-wide and has been shown to induce neutralizing antibodiesthat last as long as 35 years after a single immunization. Theirrecent studies (91) showed that YF-17D activates multiple sub-sets of DCs by signalling through multiple TLRs, includingTLR2, 7, 8 and 9 (91). The activation of multiple subsets ofDCs resulted in diverse types of adaptive immune responses(92). Thus, triggering multiple TLRs may generate immune




synergy. Based on these types of investigations, future vaccinesmay consist of multiple TLR ligands that provide both immunesynergy and immune diversity.

CONCLUSIONSThe innate immune system is evolutionarily ancient and pro-vides our first line of defense against infections. We haverecently become aware that the innate immune system usesmultiple PRR families to detect highly conserved PAMPs.Overall, the innate system is poised to protect all avenues ofpathogen invasion, with TLRs detecting pathogens at the cel-lular plasma membrane and in endosomal/lysosomal cellularcompartments, while NOD proteins and RNA helicases detectinfection in the cytoplasm. The variety of PRR families per-mits not only diversity of ligand recognition but also functionaldiversity with regard to induced responses. Although we are inthe early stages of understanding innate immune sensing andresponses, there is a growing amount of evidence suggestingthat the use of ligands, agonists and antagonists of innateimmunity (‘innate immunologicals’) can serve as novelapproaches to provide almost immediate protection against, ortreatment of, bacterial, viral or parasitic infections.Furthermore, the renewed recognition of the importance ofinnate immunity in imitating and shaping adaptive immuneresponses, along with an increasing number of studies showingthe effectiveness of TLR ligands and agonists as vaccine adju-vants, promises to lead to improved next-generation vaccines.

ACKNOWLEDGEMENTS: Dr Rosenthal is supported by aCareer Scientist Award from the Ontario HIV Treatment Network(OHTN). The author is grateful for the continuing commitmentand drive of some of his past and current Postdoctoral Fellows(Drs W Scott Gallichan, Nancy Dumais, Xiao-Dan Yao and MarieEstcourt), graduate students (including Dusan Sajic, AmandaKwant, Janina Jiang, Sumiti Jain, Anna Drannik and Vera Tang),and technicians (Jennifer Newton and Amy Patrick). The author isalso grateful for the collaboration of his colleagues, Drs Ali Ashkarand Charu Kaushic. This study was supported by grants from theCanadian Institutes of Health Research (CIHR), OHTN, theCanadian Network for Vaccines and Immunotherapeutics (CAN-VAC), and more recently, the Bill and Melinda Gates Foundation.

Rosenthal


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